The a4b2 subtype of the nicotinic receptor (nAChR) is the subtype most strongly associated with nicotine dependence as well as the treatment with nicotinic agonists of Alzheimer's Disease and other dementias. As a prototypical member of the ligand gated ion channel class of receptors, it initiates rapid responses to the neurotransmitter acetylcholine (ACh) by gating a cation-specific channel. Arachidonylethanolamide (AEA, anandamide) is an endogenous neurotransmitter that acts as an agnoist at cannabinoid receptors both in the periphery (CB2 receptors mostly) as well as in the central nervous system (CB1 mostly). Recently, however, this lipid-like agent has been shown to act directly at various receptors and ion channels. Our objective was to test whether AEA has direct actions at the nAChR.[unreadable] The nAChR was stably expressed in the cell line SH-EP1, obtained from R. Lucas (Phoenix, AZ). The cells were studied under whole cell voltage clamp conditions using 100% series resistance compensation. Rapid superfusion of ACh generated currents that rose rapidly to a peak (in about 80 ms) and desensitized as a double exponential function of time in the continued presence of ACh (5 s). This decay was modeled as two sequential desensitized states with forward rate constants k1 and k2 and backward rate constants k-1 and k-2. The rate constant k1 increased with the ACh response, in conformity with the sequential mechanism, while the others remained unchanged. [unreadable] When AEA was superfused onto the SH-EP1 cells, the peak response to ACh was diminished and continued to decrease with time up to about 50 min. This slow time course is expected for a lipophilic compound such as AEA. The recovery also required tens of minutes, but was accelerated to under 10 min by using the lipid scavenger bovine serum albumin. More strikingly, AEA increased k1 in direct proportion to the AEA concentration without evidence of saturation. The increase in k1, up to 25-fold at 2 micromolar AEA concentration, generated the spike-like responses to ACh. Simulations showed that the very rapid entry into desensitization caused by AEA could account for about 80% of the decrease in the peak amplitude because responses closer to the spreading ACh front desensitized before summating with the responses at a further diffusional distance. The rest of the decrease was postulated to occur through desensitization of the inactive state of the AChR.[unreadable] Confocal microscopy of the fluorescent Ca++ indicator fluro-3 showed that ACh caused transient increases in Ca++ concentration. To test if lipophilic AEA accelerated desensitization through an intracellular Ca++ dependent mechanism, we first showed that AEA dialyzed into the cell through the patch pipette had no effect on ACh currents. Next we used the fast acting Ca++ chelator BAPTA in the patch pipette and showed that it failed to oppose the AEA effects.[unreadable] To explore pharmacological specificity of the AEA effects, we showed that the cannabinoid agonist delta-9-tetrahydrocannabinol (THC) had no effects whatever at the nAChR. The CB1 antagonist SR-141716A, co-administered with AEA, failed to antagonize the AEA depression of peak amplitude, though it did antagonize the increase in k1 slightly. Finally, in a preliminary test of the structural requirements of the AEA effect, we tested the octahydro analogue, arachidoyl ethanolamide (H-8-AEA). Despite the structural similarity to AEA, H-8-AEA at 1 micromolar concentration was devoid of activity at the nAChR.[unreadable] We conclude that AEA, at physiologically relevant concentrations, directly blocks ACh responses at the nAChR primarily by greatly increasing its rate of desensitization. As this rate increased linearly with the AEA concentration, we suspect that it acts through graded alterations of the membrane. Experiments to test this hypothesis are planned.